Сибирский федеральный университет

Тип публикации: статья из журнала

Год издания: 2021

Идентификатор DOI: 10.3390/ijms22168827

Ключевые слова: bacterial luciferase, diffusion limitation, non-steady-state reaction kinetics, viscosity

Аннотация: Enzymes activity in a cell is determined by many factors, among which viscosity of the microenvironment plays a significant role. Various cosolvents can imitate intracellular conditions in vitro, allowing to reduce a combination of different regulatory effects. The aim of the study was to analyze the media viscosity effects on the rate constants of the separate stages of the bacterial biolumi-nescent reaction. Non-steady-state reaction kinetics in glycerol and sucrose solutions was measured by stopped-flow technique and analyzed with a mathematical model developed in accordance with the sequence of reaction stages. Molecular dynamics methods were applied to reveal the effects of cosolvents on luciferase structure. We observed both in glycerol and in sucrose media that the stages of luciferase binding with flavin and aldehyde, in contrast to oxygen, are diffusion-limited. More-over, unlike glycerol, sucrose solutions enhanced the rate of an electronically excited intermediate formation. The MD simulations showed that, in comparison with sucrose, glycerol molecules could penetrate the active-site gorge, but sucrose solutions caused a conformational change of functionally important αGlu175 of luciferase. Therefore, both cosolvents induce diffusion limitation of substrates binding. However, in sucrose media, increasing enzyme catalytic constant neutralizes viscosity effects. The activating effect of sucrose can be attributed to its exclusion from the catalytic gorge of luciferase and promotion of the formation of the active site structure favorable for the catalysis. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Журнал: International Journal of Molecular Sciences

Выпуск журнала: Vol. 22, Is. 16

Номера страниц: 8827

ISSN журнала: 16616596

Издатель: MDPI AG

• Lisitsa Albert E. (Siberian Fed Univ, Biophys Dept, Svobodny 79, Krasnoyarsk 660041, Russia)
• Sukovatyi Lev A. (Siberian Fed Univ, Biophys Dept, Svobodny 79, Krasnoyarsk 660041, Russia)
• Bartsev Sergey (Siberian Fed Univ, Biophys Dept, Svobodny 79, Krasnoyarsk 660041, Russia; Inst Biophys SB RAS, Akad Gorodok 50-50, Krasnoyarsk 660036, Russia)
• Deeva Anna A. (Siberian Fed Univ, Biophys Dept, Svobodny 79, Krasnoyarsk 660041, Russia)
• Kratasyuk Valentina A. (Siberian Fed Univ, Biophys Dept, Svobodny 79, Krasnoyarsk 660041, Russia; Inst Biophys SB RAS, Akad Gorodok 50-50, Krasnoyarsk 660036, Russia)
• Nemtseva Elena (Siberian Fed Univ, Biophys Dept, Svobodny 79, Krasnoyarsk 660041, Russia; Inst Biophys SB RAS, Akad Gorodok 50-50, Krasnoyarsk 660036, Russia)

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